Ophthalmic products intended for repeated use after opening, that is “multi-dose” products, must be preserved to minimize contamination with microorganisms during use. Preservatives that are used in ophthalmic solutions are often irritating to the eye, and at worst, may damage eye tissue after repeated use. Preservative problems may be worsened in contact lens solutions when a contact lens that has been exposed to a preservative in a lens care solution acts as a reservoir that concentrates the preservative in the eye.
In the United States, acceptably preserved pharmaceutical products, including ophthalmic, nasal and otic preparations, must achieve minimum performance standards when tested according to the procedures of the United States Pharmacopoeia Preservative Efficacy Test (PET). According to the PET protocol, adequately preserved formulations must): a) reduce 0 day challenge inocula of the bacteria Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli by at least 99.9% (3 logs) within 14 days after the challenge date; b) prevent growth of Aspergillus niger and Candida albicans within 14 days following the 0 day challenge; and c) prevent growth of the test microorganisms beyond the 14 day levels up to day 28. To demonstrate preservative efficacy for contact lens care products, a modified PET procedure is required by the FDA wherein a re-challenge of the test solutions is done on day 14 after the 14 day organism concentrations are determined.
Chitosan, the de-acetylation product of chitin, is a non-toxic biopolymer with weak antimicrobial activity. Heretofore, the use of chitosan to preserve pharmaceutical compositions has been hampered by its insolubility at pH above 6 and also because the antimicrobial activity of Chitosan in acidic solutions, by itself, is too low to meet PET requirements. Chitosan's water solubility at near neutral pH can be improved by derivatization with hydrophilic functional groups, such as carboxymethyl or glycol substituents, or by selective N-acetylation of commercially available chitosans.
Considerable efforts have been made to extend the water solubility of chitosan at neutral pH. In Sannan et al., Makromol Chem. 177, 3589 (1976), it was reported that, by treatment of chitin with alkali under homogeneous conditions, chitin with about 50% deacetylation became water-soluble. However, long reaction time and large quantities of solvent are required in some stages, including neutralization of the reaction mixture and removal of the resulting salt. This laborious process would be troublesome especially in large-scale production.
Kurita et al., Carbohydrate Polymers 16, 83 (1991), also discloses preparing water-soluble chitosan with about 50% N-acetylation by acetylating a 90% deacetylated chitosan with a complex solvent system, comprising aqueous acetic acid/methanol/pyridine. Kurita et al. describes that the resultant partially N-acetylated chitosan is water soluble, if the degree of acetylation is controlled at 50% and the acetyl groups are distributed randomly. However, the huge excess of pyridine solvent used by the Kurita method made this process impractical. Furthermore, the reaction products have limited water solubility at neutral pH because heterogeneous reaction conditions were employed that restrict uniform, random acetylation. Specifically, Kurita's chitosan reactant was not soluble in the reaction mixture, but instead it was dispersed as a swollen gel which hindered complete availability of reaction sites. In this case, the acetylation reaction would be favored in those chain segments that were most exposed and free to the reaction mixture, while other parts of the gel would be comparatively less acetylated due to steric interference from adjacent polymer chain segments. When taken as a whole, the polymer chain is not uniformly random, but instead is comprised of blocks of higher and lower acetylation.
Kubota et al., Polymer Journal. 29, 123 (1997), reported to have a facile preparation of water-soluble N-acetylated chitosan. In this reference, the chitosan is degraded by treatment with NaBO3, and the depolymerized product is then N-acetylated with acetic anhydride in aqueous acetic acid. Since both physical-chemical and biological properties of chitosan are dependent upon the chemistry of the polymer, such as the random distribution of a definite amount of acetyl groups and the molecular weight of the polymer, this process, which involves depolymerization, might alter the biological properties of chitosan.